Factors influencing the efficiency of an agroinjection-mediated SCMV-based systemic heterologous gene expression system in maize

Document Type : Research Paper

Authors

1 University of Zabol

2 Department of Biotechnology, Faculty of Agriculture, University of Zabol

3 Associate professor, Department of Biotechnology, Faculty of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran

4 Associate professor, Department of Plant Breeding and Biotechnology, Faculty of Agriculture, University of Zabol, Zabol, Iran.

5 Boyce Thompson Institute for Plant Research

Abstract

Objective
Maize (Zea mays L.) is a key cereal crop throughout the world and important model for plant genetics and biology. Establishment of an efficient transient gene expression system in maize facilitates plant functional genomics projects using gene silencing or heterologous protein overexpression strategies. The present study was aimed at optimizing an Agroinjection-mediated SCMV-based systemic heterologous gene expression system in maize.
Materials and methods
Recombinant DNA encoding sugarcane mosaic virus (SCMV) containing the coding sequence of green fluorescent protein between the protein 1 (P1) and helper component‐proteinase (HC‐Pro) cistrons, in‐frame with the viral open reading frame, was introduced into the meristematic tissue, above the coleoptilar node, of maize seedlings via a direct Agroinoculation procedure. The efficiency of Agrobacterium tumefaciens strains EHA105 and GV3101 in delivering the recombinant vector into three and seven-day old seedlings of three maize varieties, including sweet corn (Iochief and Golden Bantam varieties) and dent corn (inbred line B73), was examined. Expression of GFP transgene in symptomatic Agroinoculated plants was assessed by confocal fluorescent microscopy and RT-PCR in comparison with controls.
Results
Results of RT-PCR and confocal fluorescent microscopy revealed that: 1) A. tumefaciens GV3101 is significantly more successful in delivering the recombinant SCMV-based vector into maize plants than EHA105, 2) the percentage of GFP-expressing Golden Bantam plants is significantly higher than two other maize varieties, and 3) The effectiveness of growth stage of maize seedlings on the percentage of GFP expressing Agroinjected plants depends upon the interactions between Agrobacterium strain and maize genotype.
Conclusions
In conclusion, a combination of the Golden Bantam maize variety and A. tumefaciens strain GV3101 for direct Agroinjection of the SCMV-based vector into seedlings at the early two-leaf stage could be a fast and efficient system for investigating gene functions in maize.

Keywords

References

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Agricultural Biotechnology Journal
Volume 13, Issue 4
January 2022
Pages 159-182

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